This invention relates to methods, particularly mass spectrometric methods, for the analysis and sequencing of nucleic acid molecules.
Since the recognition of nucleic acid as the carrier of the genetic code, a great deal of interest has centered around determining the sequence of that code in the many forms in which it occurs. Two studies made the process of nucleic acid sequencing, at least with DNA, a common and relatively rapid procedure practiced in most laboratories. The first describes a process whereby terminally labeled DNA molecules are chemically cleaved at single base repetitions (A. M. Maxam and W. Gilbert, Proc. Natl. Acad. Sci. USA 74:560-64, 1977). Each base position in the nucleic acid sequence is then determined from the molecular weights of fragments produced by partial cleavage. Individual reactions were devised to cleave preferentially at guanine, at adenine, at cytosine and thymine, and at cytosine alone. When the products of these four reactions are resolved by molecular weight, using, for example, polyacrylamide gel electrophoresis, DNA sequences can be read from the pattern of fragments on the resolved gel.
In another method DNA is sequenced using a variation of the plus-minus method (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74:5463-67, 1977). This procedure takes advantage of the chain terminating ability of dideoxynucleoside triphosphates (ddNTPs) and the ability of DNA polymerase to incorporate ddNTPs with nearly equal fidelity as the natural substrate of DNA polymerase, deoxynucleoside triphosphates (dNTPs). Briefly, a primer, usually an oligonucleotide, and a template DNA are incubated in the presence of a useful concentration of all four dNTPs plus a limited amount of a single ddNTP. The DNA polymerase occasionally incorporates a dideoxynucleotide that terminates chain extension. Because the dideoxynucleotide has no 3xe2x80x2-hydroxyl, the initiation point for the polymerase enzyme is lost. Polymerization produces a mixture of fragments of varied sizes, all having identical 3xe2x80x2 termini. Fractionation of the mixture by, for example, polyacrylamide gel electrophoresis, produces a pattern that indicates the presence and position of each base in the nucleic acid. Reactions with each of the four ddNTPs permits the nucleic acid sequence to be read from a resolved gel.
These procedures are cumbersome and are limited to sequencing DNA. In addition, with conventional procedures, individual sequences are separated by, for example, electrophoresis using capillary or slab gels, which slow. Mass spectrometry has been adapted and used for sequencing and detection of nucleic acid molecules (see, e.g., U.S. Pat. Nos. 6,194,144; 6,225,450; 5,691,141; 5,547,835; 6,238,871; 5,605,798; 6,043,031; 6,197,498; 6,235,478; 6,221,601; 6,221,605). In particular, Matrix-Assisted Laser Desorption/Ionization (MALDI) and ElectroSpray Ionization (ESI), which allow intact ionization, detection and exact mass determination of large molecules, i.e. well exceeding 300 kDa in mass have been used for sequencing of nucleic acid molecules.
A further refinement in mass spectrometric analysis of high molecular weight molecules was the development of time of flight mass spectrometry (TOF-MS) with matrix-assisted laser desorption ionization (MALDI). This process involves placing the sample into a matrix that contains molecules that assist in the desorption process by absorbing energy at the frequency used to desorb the sample. Time of flight analysis uses the travel time or flight time of the various ionic species as an accurate indicator of molecular mass. Due to its speed and high resolution, time-of-flight mass spectrometry is well-suited to the task of short-range, i.e., less than 30 base sequencing of nucleic acids. Since each of the four naturally occurring nucleotide bases dC, dT, dA and dG, also referred to herein as C, T, A and G, in DNA has a different molecular weight,
MC=289.2
MT=304.2
MA=313.2
MG=329.2,
where MC, MT, MA, MG are average molecular weights in daltons of the nucleotide bases deoxycytidine, thymidine, deoxyadenosine, and deoxyguanosine, respectively, it is possible to read an entire sequence in a single mass spectrum. If a single spectrum is used to analyze the products of a conventional Sanger sequencing reaction, where chain termination is achieved at every base position by the incorporation of dideoxynucleotides, a base sequence can be determined by calculation of the mass differences between adjacent peaks. In addition, the method can be used to determine the masses, lengths and base compositions of mixtures of oligonucleotides and to detect target oligonucleotides based upon molecular weight.
MALDI-TOF mass spectrometry for sequencing DNA using mass modification (see, e.g., U.S. Pat. Nos. 5,547,835, 6,194,144; 6,225,450; 5,691,141 and 6,238,871) to increase mass resolution is available. The methods employ conventional Sanger sequencing reactions with each of the four dideoxynucleotides. In addition, for example for multiplexing, two of the four natural bases are replaced; dG is substituted with 7-deaza-dG and dA with 7-deaza-dA.
A variety of techniques and combinations thereof have been directed to improving the level of accuracy in determining the nucleotide compositions of mixtures of oligonucleotides using mass spectrometry, and many of these methods employ nucleotide analogs. For example, Muddiman et al. (Anal. Chem., 69(8): 1543-1549, 1997) discusses an algorithm for the unique definition of the base composition of PCR-amplified products, especially longer ( greater than 100 bp) oligonucleotides. The algorithm places a constraint on the otherwise large number of possible base compositions for long oligonucleotides by taking into account only those masses (measured by electrospray ionization mass spectrometry) that are consistent with that of their denatured complementary strands, assuming Watson-Crick base-pairing. In addition, the algorithm imposes the constraint of known primer compositions, since the primer sequences are known, and this constraint becomes especially significant with shorter PCR products whose mass of xe2x80x9cunknownxe2x80x9d sequence relative to that of the primer mass is small. Muddiman et al. also discusses invoking additional measurements for defining the base composition with even greater accuracy. These include the possibility of post-modifying the PCR product using e.g., dimethyl sulfate to selectively methylate every xe2x80x9cGxe2x80x9d in the PCR product, or using a modified base during PCR amplification, conducting mass measurements on the modified oligonucleotides, and comparing the mass measurements with those of the unmodified complementary strands.
Chen et al. (Anal. Chem., 71(15): 3118-3125, 1999) reports a method that combines stable isotope 13C/15N labelling of PCR products with analysis of the mass shifts by MALDI-TOF mass spectrometry. The mass shift due to labelling of a single type of nucleotide (i.e, A, T, G or C) reveals the number of that type of nucleotide in a given fragment. While the method is useful in the measurement and comparison of nucleotide compositions of homologous sequences for sequence validation and in scoring polymorphisms, tedious repetitive sequencing reactions (using the four different labelled nucleotides) and mass spectrometric measurements are required.
Hence there is a need in the art for methods that (i) unambiguously assign nucleotides in a sequence, and, (ii) resolve large numbers of oligonucleotides that have the same length, different base compositions, and nearly equal (i.e., less than or equal to about 1 dalton difference) molecular weights. Therefore it is an object herein to provide methods that solve such problems
Provided herein are methods for sequencing and detecting nucleic acids using techniques, such as mass spectrometry and gel electrophoresis, that are based upon molecular mass. The methods use deoxynucleotide analogs, modified nucleotide terminators and/or mass-labeled primers in one or more reactions for sequencing or detection protocols that involve primer extension, and analyze these products from more than one oligonucleotide on, for example, a single mass spectrum. This provides a means for accurate detection and/or sequencing of a an oligonucleotide and is particularly advantageous for detecting or sequencing a plurality target nucleic acid molecules in a single reaction using any technique that distinguishes products based upon molecular weight. The methods herein are particularly adapted for mass spectrometric analyses.
For example, a sequencing method provided herein uses deoxynucleotide analogs, modified nucleotide terminators and/or mass-labeled primers in one or more Sanger sequencing reactions, and analyzes these products from more than one oligonucleotide on a single mass spectrum. In particular, a plurality of primers can be used to simultaneously sequence a plurality of nucleic acid molecules or portions of the same molecule. By extending the primers with mass-matched nucleotides, the resulting products mass shifts that are periodically related to the size of the original primer.
As a result, the sequence of any given oligonucleotide can be determined with a high level of accuracy, and also mixtures of a number of sequences can be multiplexed in a single mass spectrum. The limit on the number of oligonucleotides that can be sequenced simultaneously is governed by the base periodicity, the maximum mass shift, and the resolving power of analytical tool, such as the mass spectrometer. The base periodicity and maximum mass shift can be carefully engineered for optimal resolution and accuracy, depending on the number of sequences to be simultaneously analyzed, and the information desired; as many sequences as desired can be sequenced simultaneously especially in the detection and scoring of single nucleotide polymorphisms, insertions, deletions and other mutations.
In another embodiment, a target nucleic acid molecule is sequenced using mass-matched nucleotides and chain terminating nucleotides. For example, a primer is annealed to a target nucleic acid, the primer is extended in the presence of chain-terminating nucleotides and mass-matched nucleotides to produce extension products, the masses of the extension products follow a periodic distribution that is determined by the mass of the mass-matched nucleotides, and the sequence of the target nucleic acid is determined from the mass shift of each extension product from its corresponding periodic reference mass by virtue of incorporation of the chain terminator. The mass-matched nucleotides all have identical masses, and each chain terminating nucleotide has a distinct mass that differs from that of the other chain terminating nucleotides. This results in unique predetermined values of mass shift corresponding to each chain terminating nucleotide and based upon the original primer.
This method is adaptable for any sequencing method or detection method that relies upon or includes chain extension. These methods include, but are not limited to, sequencing methods based upon Sanger sequencing, and detection methods, such as primer oligo base extension (PROBE) (see, e.g., U.S. Pat. No. 6,043,031; allowed U.S. application Ser. No. 09/287,679; and U.S. Pat. No. 6,235,478), that rely include a step of chain extension.
Also, contemplated are methods, such as haplotyping methods, in which two mutations in the same gene are detection are provided. A detector (primer) oligonucleotide is to the hybridized to the first mutation and the primer is extended with mass-matched nucleotides and appropriately selected chain terminator(s) to detect the second mutation.
In other embodiments, a plurality of target nucleic acids can be multiplexed in a single reaction measurement by annealing each target nucleic acid to a primer of distinct molecular weight each primer is then extended with mass-matched nucleotides and chain terminators in formats that depend upon whether detection or sequencing is desired. These methods are particularly useful for methods of detection in which a primer is hybridized to a plurality of target nucleic acid molecules, such as immobilized nucleic acid molecules, hybrids separated from unhybridized nucleic acids and the detectors detected. Such methods include PROBE, in which case the extension reaction is performed in the presence chain terminators and mass matched deoxynucleotides.
The primers of distinct molecular weight can be selected to differ in molecular weight by a value that is greater than the maximum mass shift, i.e., the difference in molecular weight between the heaviest and the lightest nucleotide terminators in chain extension reactions. The difference in molecular weight between the primers for a plurality of target nucleic acids can be selected to be least 20 daltons greater than the maximum mass shift to account for the finite band width of the peaks.
The number of molecules that can be multiplexed is governed by the periodicity, the maximum mass shift, and the resolving power of the sequence detection instrument. In some embodiments, about 7 to about 25 or more molecules can be multiplexed. For scoring single nucleotide polymorphisms, only a single nucleotide terminator is required (depending on the base identity of the single nucleotide polymorphism). In this case, the maximum mass shift required is identically zero, so that larger numbers of molecules, greater than 25, 35, 50 and more, can be multiplexed, depending on the resolving power of the sequencing format, and for mass spectrometry the instrument. Depending on the amount of sequence information desired, one, two or three rather than four types of nucleotide terminators (corresponding to each of the four nucleic acid bases) can be used.
In other embodiments, the mass shift is obtained using pair-matched nucleotides, i.e., the mass of each nucleotide base-pair is selected so that the masses of all pairs are identical. In one embodiment thereof, the following steps are performed: (i) the target nucleic acid is copied or amplified by a method such as PCR in the presence of the pair-matched nucleotide set prior to the sequencing or detection reaction; (ii) the target nucleic acid is denatured, and a partially duplex hairpin primer is annealed and ligated to the single-stranded template; (iii) the primer is extended in the presence of chain terminating nucleotides and pair-matched nucleotides to produce extension products, where the masses of the extension products follow a periodic distribution that is determined by the mass of the pair-matched nucleotide set, and, (iv) the target nucleic acid is detected by virtue of its molecular weight or its sequence is determined from the mass shift of each extension product from its corresponding periodic reference mass.
In another embodiment, the mass of each terminating base pair is unique and resolvable, so that the mass shifts corresponding to each terminating base pair are unique. The nucleotide terminators are optionally mass-matched or can be of distinct masses as long as distinct values of mass shift are obtained for each terminating base pair.
In another embodiment, the extension products are treated to produce blunt-ended double-stranded extension products by methods known to those of skill in the art, such as the use of single-strand specific nucleases. In an aspect of this embodiment, a plurality of target nucleic acids can be multiplexed in a single reaction by annealing each target nucleic acid to a primer of distinct molecular weight. The primers can be selected to differ in molecular weight by a value that is greater than the maximum mass shift, i.e., the difference in molecular weight between the heaviest and the lightest nucleotide terminating base pairs. Since double stranded nucleic acid can be analyzed, the effective sequence read is halved relative to the embodiment employing mass-matched nucleotides, but the number of molecules that can be multiplexed is doubled, due to the increase in period (the value of the mass of a base pair, rather than a single mass-matched nucleotide). In exemplary embodiments, about 14 to about 50 sequences are multiplexed. In detection embodiments, about 50 or more molecules can be simultaneously multiplexed since only a single terminating base pair is added in the extension reaction.
In another embodiment, the chain termination reactions are carried out separately using a standard nucleotide terminator, pair-matched nucleotides, and mass-labeled primers, if modified nucleotide terminators which are either mass-matched or provide distinct values of mass shift for each terminating base pair are not available. The reactions are pooled prior to detection or sequence analysis. In one embodiment, the mass-labeled primers can have distinct values of molecular weight that give rise to unique values of mass shift or positional mass difference for each terminating base.
In andother method provided herein, a population of nucleic acids having the same length but different base compositions can be resolved by synthesizing the nucleic acids in the presence of a nucleotide analog to produce synthesized nucleic acids having incorporated the nucleotide analog, where the nucleotide analog is selected to optimally separate the masses of the population of nucleic acids according to their individual base compositions. For example, the nucleotide analog or analogs are selected to separate the population of nucleic acids according to base composition by greater than 1 dalton. In another embodiment, the nucleotide analog or analogs are selected to separate the population of nucleic acids according to base composition by mass values of about 3 daltons to about 8 daltons, depending on the choice of analog and on the resolving power of the detection instrument. In other embodiments, the nucleotide analog or analogs can be selected to restrict oligonucleotides having the same length to have the same mass, i.e., a peak separation of zero, regardless of differences in base composition, such as in detection methods, where it is desirable to separate populations of oligonucleotides according to their length.
Nucleic acid molecules that contain mass-matched nucleotides and/or pair-matched nucleotides are provided.
Also provided are combinations for practicing the methods provided herein. For instance, in one embodiment, the combinations include a set of mass-matched deoxynucleotides. In another embodiment, the combinations a set of pair-matched nucleotides and a set of mass-matched chain terminating nucleotides. In another embodiment, the combination includes a set of pair-matched nucleotides and chain terminating nucleotides which form terminating base pairs of distinctly different molecular weight. In yet another embodiment, the combination includes a set of pair-matched nucleotides and mass-labeled primers. In other embodiments, mass-staggered primers can be added to as optional components.
Kits containing the combinations with optional instructions and/or additional reagents are also provided. The kits contain the reagents as described herein and optionally any other reagents required to perform the reactions. Such reagents and compositions are packaged in standard packaging known to those of skill in the art. Additional vials, containers, pipets, syringes and other products for sequencing can also be included. Instructions for performing the reactions can be included.
Also provided herein are methods for optimization of the analysis of base compositions of mixtures of oligonucleotides by mass spectrometry. A single spectrum can be used to resolve a very large number of oligonucleotides having the same length but different molecular weights by incorporating a nucleotide analog into the oligonucleotides in the mixture such that the peaks are no closer than a minimum value called peak separation. The peak separation can be tailored by careful selection of the nucleotide analog and of a mass spectrometer with the desired resolving power.
The methods herein permit unambiguous and accurate analysis of the sequences or molecular weights of large numbers of oligonucleotides in a single mass spectrum by combining the rapidity of mass spectrometry with the resolving power of nucleotide analogs which are carefully selected and incorporated into the oligonucleotide mixture according to the desired application.
Other features and advantages will be apparent from the following detailed description and claims.